Human protein kinase H2LAU20

ABSTRACT

The H2LAU20 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing H2LAU20 polypeptides and polynucleotides in therapy, and diagnostic assays for such.

This application is a division of application Ser. No. 09/126,646, filedJul. 31, 1998, now U.S. Pat. No. 6,001,623 whose contents areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to newly identified polypeptides andpolynucleotides encoding such polypeptides, to their use in therapy andin identifying compounds which may be agonists, antagonists and/orinhibitors which are potentially useful in therapy, and to production ofsuch polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

The drug discovery process is currently undergoing a fundamentalrevolution as it embraces ‘functional genomics’, that is, highthroughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on ‘positional cloning’. Aphenotype, that is a biological function or genetic disease, would beidentified and this would then be tracked back to the responsible gene,based on its genetic map position.

Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available. There is a continuingneed to identify and characterise further genes and their relatedpolypeptides/proteins, as targets for drug discovery.

A number of polypeptide growth factors and hormones mediate theircellular effects through a signal transduction pathway. Transduction ofsignals from the cell surface receptors for these ligands tointracellular effectors frequently involves phosphorylation ordephosphorylation of specific protein substrates by regulatory proteinserine/threonine kinases (PSTK) and phosphatases. Serine/threoninephosphorylation is a major mediator of signal transduction inmulticellular organisms. Receptor-bound, membrane-bound andintracellular PSTKs regulate cell proliferation, cell differentiationand signalling processes in many cell types.

Aberrant protein serine/threonine kinase activity has been implicated oris suspected in a number of pathologies such as rheumatoid arthritis,psoriasis, septic shock, bone loss, many cancers and other proliferativediseases. Accordingly, serine/threonine kinases and the signaltransduction pathways which they are part of are potential targets fordrug design.

A subset of PSTKs are involved in regulation of cell cycling. These arethe cyclin-dependent kinases or CDKs (Peter and Herskowitz, Cell 1994:79, 181-184). CDKs are activated by binding to regulatory proteinscalled cyclins and control passage of the cell through specific cellcycle checkpoints. For example. CDK2 complexed with cyclin E allowscells to progress through the G1 to S phase transition. The complexes ofCDKs and cyclins are subject to inhibition by low molecular weightproteins such as p16 (Serrano et al, Nature 1993: 366, 704), which bindsto and inhibits CDK4. Deletions or mutations in p16 have been implicatedin a variety of tumors (Kamb et al, Science 1994: 264, 436-440).Therefore, the proliferative state of cells and diseases associated withthis state are dependent on the activity of CDKs and their associatedregulatory molecules. In diseases such as cancer where inhibition ofproliferation is desired, compounds that inhibit CDKs may be usefultherapeutic agents. Conversely, activators of CDKs may be useful whereenhancement of proliferation is needed, such as in the treatment ofimmunodeficiency.

Frequently, in diseases such as osteoporosis and osteoarthritis,patients have established lesions of bone or cartilage, respectively.Treatment of such lesions requires an agent that will stimulate new boneor cartilage formation to replace that lost to the disease; therefore,there is a need for drugs that increase the number of osteoblasts orchondrocytes, the cells responsible for bone or cartilage formation,respectively. Similarly, replacement of heart or skeletal muscledepleted by diseases such as myocardial infarction or HIV-associatedcachexia requires drugs that stimulate proliferation of cardiac myocytesor skeletal myoblasts. The present invention describes a novel humanclone, H2LAU20, which shares homology with predicted PSTK's from S.pombe, D. melanogaster, C. elegans, and S. cerevisiae and has motifsassociated with other known protein kinases. Inhibitors of H2LAU20 areexpected to regulate proliferation of cell growth.

SUMMARY OF THE INVENTION

The present invention relates to H2LAU20, in particular H2LAU20polypeptides and H2LAU20 polynucleotides, recombinant materials andmethods for their production. In another aspect, the invention relatesto methods for using such polypeptides and polynucleotides, includingthe treatment of bone loss including osteoporosis; inflammatory diseasessuch as Adult Respiratory Disease Syndrome (ARDS), Rheumatoid arthritis,Osteoarthritis, Inflammatory Bowel Disease (IBD), psoriasis, dermatitis,asthma, allergies; diabetes and associated disorders; infections such asbacterial, fungal, protozoan and viral infections, particularlyinfections caused by HIV-1 or HIV-2; HIV-associated cachexia and otherimmunodeficiency disorders; septic shock; pain; injury; cancersincluding testicular cancer; anorexia; bulimia: Parkinson's disease;cardiovascular diseases including restenosis, atherosclerosis, acuteheart failure. myocardial infarction; hypotension; hypertension; urinaryretention; angina pectoris; ulcers; benign prostatic hypertrophy; andpsychotic and neurological disorders, including anxiety, schizophrenia,manic depression, delirium, dementia, severe mental retardation anddyskinesias, such as Huntington's disease or Gilles de la Tourette'ssyndrome, hereinafter referred to as “the Diseases”, amongst others. Ina further aspect, the invention relates to methods for identifyingagonists and antagonists/inhibitors using the materials provided by theinvention, and treating conditions associated with H2LAU20 imbalancewith the identified compounds. In a still further aspect, the inventionrelates to diagnostic assays for detecting diseases associated withinappropriate H2LAU20 activity or levels.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to H2LAU20polypeptides. Such peptides include isolated polypeptides comprising anamino acid sequence which has at least 70% identity, preferably at least80% identity, more preferably at least 90% identity, yet more preferablyat least 95% identity, most preferably at least 97-99% identity, to thatof SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptidesinclude those comprising the amino acid of SEQ ID NO:2.

Further peptides of the present invention include isolated polypeptidesin which the amino acid sequence has at least 70% identity, preferablyat least 80% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, most preferably at least 97-99%identity, to the amino acid sequence of SEQ ID NO:2 over the entirelength of SEQ ID NO:2. Such polypeptides include the polypeptide of SEQID NO:2.

Further peptides of the present invention include isolated polypeptidesencoded by a polynucleotide comprising the sequence contained in SEQ IDNO: 1.

Polypeptides of the present invention are believed to be members of theserine/threonine kinase family of polypeptides. They are therefore ofinterest because aberrant protein serine/threonine kinase activity hasbeen implicated or is suspected in a number of pathologies such asrheumatoid arthritis, psoriasis, septic shock, bone loss, many cancersand other proliferative diseases. Accordingly, serine/threonine kinasesand the signal transduction pathways which they are part of arepotential targets for drug design. These properties are hereinafterreferred to as “H2LAU20 activity” or “H2LAU20 polypeptide activity” or“biological activity of H2LAU20. Also included amonast these activitiesare antigenic and immunogenic activities of said H2LAU20 polypeptides,in particular the antigenic and immunogenic activities of thepolypeptide of SEQ ID NO:2. Preferably, a polypeptide of the presentinvention exhibits at least one biological activity of H2LAU20.

The polypeptides of the present invention may be in the form of the“mature” protein or may be a part of a larger protein such as a fusionprotein. It is often advantageous to include an additional amino acidsequence which contains secretory or leader sequences, pro-sequences,sequences which aid in purification such as multiple histidine residues,or an additional sequence for stability during recombinant production.

The present invention also includes variants of the aforementionedpolypeptides, that is polypeptides that vary from the referents byconservative amino acid substitutions, whereby a residue is substitutedby another with like characteristics. Typical such substitutions areamong Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr. Particularly preferredare variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids aresubstituted, deleted, or added in any combination.

Polypeptides of the present invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

In a further aspect, the present invention relates to H2LAU20polynucleotides. Such polynucleotides include isolated polynucleotidescomprising a nucleotide sequence encoding a polypeptide which has atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, to theamino acid sequence of SEQ ID NO:2, over the entire length of SEQ IDNO:2. In this regard, polypeptides which have at least 97% identity arehighly preferred, whilst those with at least 98-99% identity are morehighly preferred, and those with at least 99% identity are most highlypreferred. Such polynucleotides include a polynucleotide comprising thenucleotide sequence contained in SEQ ID NO:1 encoding the polypeptide ofSEQ ID NO:2.

Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence that has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity,to a nucleotidesequence encoding a polypeptide of SEQ ID NO:2, over the entire codingregion. In this regard, polynucleotides which have at least 97% identityare highly preferred, whilst those with at least 98-99% identity aremore highly preferred, and those with at least 99% identity are mosthighly preferred.

Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence which has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, to SEQ ID NO:1 overthe entire length of SEQ ID NO:1 In this regard, polynucleotides whichhave at least 97% identity are highly preferred, whilst those with atleast 98-99% identity are more highly preferred, and those with at least99% identity are most highly preferred. Such polynucleotides include apolynucleotide comprising the polynucleotide of SEQ ID NO:1 as well asthe polynucleotide of SEQ ID NO:1.

The invention also provides polynucleotides which are complementary toall the above-described polynucleotides.

The nucleotide sequence of SEQ ID NO:1 shows homology with Human proteinkinase YAK1 (U.S. Ser. No. 08/802,466), and Human protein kinase YAK3(U.S. Ser. No. 08/835,170). The nucleotide sequence of SEQ ID NO:1 is acDNA sequence and comprises a polypeptide encoding sequence (nucteotide1 to 1863) encoding a polypeptide of 620 amino acids, the polypeptide ofSEQ ID NO:2. The nucleotide sequence encoding the polypeptide of SEQ IDNO:2 may be identical to the polypeptide encoding sequence contained inSEQ ID NO:1 or it may be a sequence other than the one contained in SEQID NO:1, which, as a result of the redundancy (degeneracy) of thegenetic code, also encodes the polypeptide of SEQ ID NO:2. Thepolypeptide of SEQ ID NO:2 is structurally related to other proteins ofthe serine/threonine kinase family, having homology and/or structuralsimilarity with C. elegans protein kinase F49E11.1 (Wilson et al.,Nature 368: 32-38, 1994) and Human protein kinase YAK1 (U.S. Ser. No.08/802,466).

Preferred polypeptides and polynucleotides of the present invention areexpected to have, inter alia, similar biological functions/properties totheir homologous polypeptides and polynucleotides. Furthermore,preferred polypeptides and polynucleotides of the present invention haveat least one H2LAU20 activity.

The present invention also relates to partial or other polynucleotideand polypeptide sequences which were first identified prior to thedetermination of the corresponding full length sequences of SEQ ID NO:1and SEQ ID NO:2.

Accordingly, in a further aspect, the present invention provides for anisolated polynucleotide comprising:

(a) a nucleotide sequence which has at least 70% identity, preferably atleast 80% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, even more preferably at least 97-99%identity to SEQ ID NO:3 over the entire length of SEQ ID NO:3;

(b) a nucleotide sequence which has at least 70% identity, preferably atleast 80% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, even more preferably at least 97-99%identity, to SEQ ID NO:3 over the entire length of SEQ ID NO:3;

(c) the polynucleotide of SEQ ID NO:3; or

(d) a nucleotide sequence encoding a polypeptide which has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% identity, to the amino acid sequence of SEQID NO:4, over the entire length of SEQ ID NO:4;

as well as the polynucleotide of SEQ ID NO:3.

The present invention further provides for a polypeptide which:

(a) comprises an amino acid sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to that of SEQ ID NO:4 over the entire length of SEQ IDNO:4;

(b) has an amino acid sequence which is at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to the amino acid sequence of SEQ ID NO:4 over theentire length of SEQ ID NO:4;

(c) comprises the amino acid of SEQ ID NO:4; and

(d) is the polypeptide of SEQ ID NO:4;

as well as polypeptides encoded by a polynucleotide comprising thesequence contained in SEQ ID NO:3.

The nucleotide sequence of SEQ ID NO:3 and the peptide sequence encodedthereby are derived from EST (Expressed Sequence Tag) sequences. It isrecognized by those skilled in the art that there will inevitably besome nucleotide sequence reading errors in EST sequences (see Adams, M.D. et al, Nature 377 (supp) 3, 1995). Accordingly, the nucleotidesequence of SEQ ID NO:3 and the peptide sequence encoded therefrom aresubject to the same inherent limitations in sequence accuracy.Furthermore, the peptide sequence encoded by SEQ ID NO:3 comprises aregion of identity or close homology and/or close structural similarity(for example a conservative amino acid difference) with the closesthomologous or structurally similar protein.

Polynucleotides of the present invention may be obtained, using standardcloning and screening techniques, from a cDNA library derived from mRNAin cells of human placenta, using the expressed sequence tag (EST)analysis (Adams, M. D., et al. Science (1991) 252:1651-1656; Adams, M.D. et al., Nature, (1992) 355:632-634; Adams, M. D., et al., Nature(1995) 377 Supp:3-174). Polynucleotides of the invention can also beobtained from natural sources such as genomic DNA libraries or can besynthesized using well-known and commercially available techniques.

When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself; or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- orprepro-protein sequence, or other fusion peptide portions. For example,a marker sequence, which facilitates purification of the fusedpolypeptide, can be encoded. In certain preferred embodiments of thisaspect of the invention, the marker sequence is a hexa-histidinepeptide, as provided in the pQE vector (Qiagen, Inc.) and described inGentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.The polynucleotide may also contain non-coding 5′ and 3′ sequences, suchas transcribed, non-translated sequences, splicing and polyadenylationsignals, ribosome binding sites and sequences that stabilize mRNA.

Further embodiments of the present invention include polynucleotidesencoding polypeptide variants which comprise the amino acid sequence ofSEQ ID NO:2 and in which several, for instance from 5 to 10, 1 to 5, 1to 3, 1 to 2 or 1, amino acid residues are substituted, deleted oradded, in any combination.

Polynucleotides which are identical or sufficiently identical to anucleotide sequence contained in SEQ ID NO:1, may be used ashybridization probes for cDNA and genomic DNA or as primers for anucleic acid amplification (PCR) reaction, to isolate full-length cDNAsand genomic clones encoding polypeptides of the present invention and toisolate CDNA and genomic clones of other genes (including genes encodinghomologs and orthologs from species other than human) that have a highsequence similarity to SEQ ID NO:1. Typically these nucleotide sequencesare 70% identical, preferably 80% identical, more preferably 90%identical, most preferably 95% identical to that of the referent. Theprobes or primers will generally comprise at least 15 nucleotides,preferably, at least 30 nucleotides and may have at least 50nucleotides. Particularly preferred probes will have between 30 and 50nucleotides.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than human, may beobtained by a process which comprises the steps of screening anappropriate library under stringent hybridization conditions with alabeled probe having the sequence of SEQ ID NO:1 or a fragment thereof;and isolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Such hybridization techniques are well known tothe skilled artisan. Preferred stringent hybridization conditionsinclude overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20microgram/ml denatured, sheared salmon sperm DNA; followed by washingthe filters in 0.1×SSC at about 65° C. Thus the present invention alsoincludes polynucleotides obtainable by screening an appropriate libraryunder stingent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or a fragment thereof.

The skilled artisan will appreciate that, in many cases, an isolatedcDNA sequence will be incomplete, in that the region coding for thepolypeptide is cut short at the 5′ end of the cDNA. This is aconsequence of reverse transcriptase, an enzyme with inherently low‘processivity’ (a measure of the ability of the enzyme to remainattached to the template during the polymerisation reaction), failing tocomplete a DNA copy of the mRNA template during 1st strand cDNAsynthesis.

There are several methods available and well known to those skilled inthe art to obtain full-length cDNAs, or extend short cDNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman et al., PNAS USA 85, 8998-9002, 1988). Recentmodifications of the technique, exemplified by the Marathon™ technology(Clontech Laboratories Inc.) for example, have significantly simplifiedthe search for longer cDNAs. In the Marathon™ technology, cDNAs havebeen prepared from mRNA extracted from a chosen tissue and an ‘adaptor’sequence ligated onto each end. Nucleic acid amplification (PCR) is thencarried out to amplify the ‘missing’ 5′ end of the cDNA using acombination of gene specific and adaptor specific oligonucleotideprimers. The PCR reaction is then repeated using ‘nested’ primers, thatis, primers designed to anneal within the amplified product (typicallyan adaptor specific primer that anneals further 3′ in the adaptorsequence and a gene specific primer that anneals further 5′ in the knowngene sequence). The products of this reaction can then be analyzed byDNA sequencing and a full-length cDNA constructed either by joining theproduct directly to the existing cDNA to give a complete sequence, orcarrying out a separate fall-length PCR using the new sequenceinformation for the design of the 5′ primer.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in the art from genetically engineered host cellscomprising expression systems. Accordingly, in a further aspect, thepresent invention relates to expression systems which comprise apolynucleotide or polynucleotides of the present invention, to hostcellswhich are genetically engineered with such expression systems and to theproduction of polypeptides of the invention by recombinant techniques.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., Basic Methods in Molecular Biology (1986)and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).Preferred such methods include, for instance, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression systems may containcontrol regions that regulate as well as engender expression. Generally,any system or vector which is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host, may be used. Theappropriate nucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al., MOLECULAR CLONING,A LABORATORY MANUAL (supra). Appropriate secretion signals may beincorporated into the desired polypeptide to allow secretion of thetranslated protein into the lumen of the endoplasmic reticulum, theperiplasmic space or the extracellular environment. These signals may beendogenous to the polypeptide or they may be heterologous signals.

If a polypeptide of the present invention is to be expressed for use inscreening assays, it is generally preferred that the polypeptide beproduced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If the polypeptide issecreted into the medium, the medium can be recovered in order torecover and purify the polypeptide. If produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

Polypeptides of the present invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Mvfost preferably, highperformnance liquid chromatography is employed for purification.Well-known techniques for refolding proteins may be employed toregenerate active conformation when the polypeptide is denatured duringisolation and or purification.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of thegene characterized by the polynucleotide of SEQ ID NO:1 which isassociated with a dysfunction will provide a diagnostic tool that canadd to, or define, a diagnosis of a disease, or susceptibility to adisease, which results from under-expression, over-expression or alteredexpression of the gene. Individuals carrying mutations in the gene maybe detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled H2LAU20 nucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations inelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing (ee, e.g., Myers et al.,Science (1985) 230:1242). Sequence changes at specific locations mayalso be revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method (see Cotton et al., Proc NatlAcad Sci USA (1985) 85: 4397-4401). In another embodiment, an array ofoligonucleotides probes comprising H2LAU20 nucleotide sequence orfragments thereof can be constructed to conduct efficient screening ofe.g., genetic mutations. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage,. and genetic variability (see for example: M.Chee et al.,Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to the Diseases through detection of mutation in theH2LAU20 gene by the methods described. In addition, such diseases may bediagnosed by methods comprising determining from a sample derived from asubject an abnormally decreased or increased level of polypeptide ormRNA. Decreased or increased expression can be measured at the RNA levelusing any of the methods well known in the art for the quantitation ofpolynucleotides, such as, for example, nucleic acid amplification, forinstance PCR, RT-PCR, RNase protection, Northern blotting and otherhybridization methods. Assay techniques that can be used to determinelevels of a protein, such as a polypeptide of the present invention, ina sample derived from a host are well known to those of skill in theart. Such assay methods include radioimmunoassays, competitive-bindingassays, Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagnostickit which comprises:

(a) a polynucleotide of the present invention, preferably the nucleotidesequence of SEQ ID NO: 1, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide of the present invention, preferably the polypeptideof SEQ ID NO:2 or a fragment thereof; or

(d) an antibody to a polypeptide of the present invention, preferably tothe polypeptide of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a disease, particularly boneloss including osteoporosis: inflammatory diseases such as AdultRespiratory Disease Syndrome (ARDS), Rheumatoid arthritis,Osteoarthritis, Inflammatory Bowel Disease (IBD), psoriasis, dermatitis,asthma, allergies; diabetes and associated disorders; infections such asbacterial, fungal, protozoan and viral infections, particularlyinfections caused by HIV-1 or HIV-2; HIV-associated cachexia and otherimmunodeficiency disorders; septic shock; pain; injury; cancersincluding testicular cancer; anorexia; bulimia; Parkinson's disease;cardiovascular disease including restenosis, atherosclerosis, acuteheart failure. myocardial infarction; hypotension; hypertension; urinaryretention; angina pectoris; ulcers; benign prostatic hypertrophy; andpsychotic and neurological disorders, including anxiety, schizophrenia,manic depression, delirium, dementia, severe mental retardation anddyskinesias, such as Huntington's disease or Gilles dela Tourett'ssyndrome, amongst others.

The nucleotide sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to, andcan hybridize with, a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound in, for example, V. McKusick, Mendelian Inheritance in Man(available on-line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them, can also be used as immunogens to produceantibodies immunospecific for polypeptides of the present invention. Theterm “immunospecific” means that the antibodies have substantiallygreater affinity for the polypeptides of the invention than theiraffinity for other related polypeptides in the prior art.

Antibodies generated against polypeptides of the present invention maybe obtained by administering the polypeptides or epitope-bearingfragments, analogs or cells to an animal, preferably a non-human animal,using routine protocols. For preparation of monoclonal antibodies, anytechnique, which provides antibodies produced by continuous cell linecultures, can be used. Examples include the hybridoma technique (Kohler,G. and Milstein, C., Nature (1975) 256:495-497), the trioma technique,the human B-cell hybridoma technique (Kozboret al., Immunology Today(1983) 4:72) and the EBV-hybridoma technique (Cole et al., MONOCLONALANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies, such as thosedescribed in U.S. Pat. No. 4,946,778, can also be adapted to producesingle chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

The above-described antibodies mav be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against polypeptides of the present invention may also beemployed to treat the Diseases, amongst others.

In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises inoculating themammal with a polypeptide of the present invention, adequate to produceantibody and/or T cell immune response to protect said animal from theDiseases hereinbefore mentioned, amongst others. Yet another aspect ofthe invention relates to a method of inducing immunological response ina mammal which comprises, delivering a polypeptide of the presentinvention via a vector directing expression of the polynucleotide andcoding for the polypeptide in vivo in order to induce such animmunological response to produce antibody to protect said animal fromdiseases.

A further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to a polypeptide of thepresent invention wherein the composition comprises a polypeptide orpolynucleotide of the present invention The vaccine formulation mayfurther comprise a suitable carrier. Since a polypeptide may be brokendown in the stomach, it is preferably administered parenterally (forinstance, subcutaneous, intramuscular, intravenous, or intradermalinjection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation instonic with the blood of the recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampoules and vials and may bestored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The vaccine formulationmay also include adjuvant systems for enhancing the immunogenicity ofthe formulation, such as oil-in water systems and other systems known inthe art. The dosage will depend on the specific activity of the vaccineand can be readily determined bv routine experimentation.

Polypeptides of the present invention are responsible for manybiological functions, including many disease states, in particular theDiseases hereinbefore mentioned. It is therefore desirous to devisescreening methods to identifv compounds which stimulate or which inhibitthe function of the polypeptide. Accordingly, in a further aspect, thepresent invention provides for a method of screening compounds toidentify those which stimulate or which inhibit the function of thepolypeptide. In general, agonists or antagonists may be employed fortherapeutic and prophylactic purposes for such Diseases as hereinbeforementioned. Compounds may be identified from a variety of sources, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. Such agonists, antagonists or inhibitors so-identifiedmay be natural or modified substrates, ligands, receptors, enzymes,etc., as the case may be, of the polypeptide; or may be structural orfunctional mimetics thereof (see Coligan et al., Current Protocols inImmunology 1(2): Chapter 5 (1991)).

The screening method may simply measure the binding of a candidatecompound to the polypeptide, or to cells or membranes bearing thepolypeptide, or a fusion protein thereof by means of a label directly orindirectly associated with the candidate compound. Alternatively, thescreening method may involve competition with a labeled competitor.Further, these screening methods may test whether the candidate compoundresults in a signal generated by activation or inhibition of thepolypeptide, using detection systems appropriate to the cells bearingthe polypeptide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptides may be employed in screening methods for inverseagonists or inhibitors, in the absence of an agonist or inhibitor, bytesting whether the candidate compound results in inhibition ofactivation of the polypeptide. Further, the screening methods may simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide of the present invention, to form a mixture,measuring H2LAU20activity in the mixture, and comparing the H2LAU20activity of the mixture to a standard. Fusion proteins, such as thosemade from Fc portion and H2LAU20 polypeptide, as hereinbefore described,can also be used for high-throughput screening assays to identifyantagonists for the polypeptide of the present invention (see D. Bennettet al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et al., JBiol Chem, 270(16):9459-9471 (1995)).

The knowledge that the H2LAU20 encodes a putative protein kinasesuggests that recombinant forms can be used to establish a proteinkinase activity. Typically, this would involve the direct incubation ofH2LAU20 with a protein or peptide substrate in the presence of 32P-ATP,followed by the measurement of radioactivity incorporated into thesubstrate by separation and counting. Separation methods includeimmunoprecipitation, conjugation of substrate to a bead allowingseparation by centrifugation or determination of incorporation byscintillation proximity assay, SDS-PAGE followed by autoradiography orbiosensor analysis. While the specific substrates are not yet known,candidates include H2LAU20 itself (autophosphorylation), myelin basicprotein, casein, histone and HSP27. Other substances might be discoveredby incubating H2LAU20 with random peptides conjugated to solid supportsor displayed on the surface of phage or by incubation of H2LAU20 withmammalian cell lysates and 32P-ATP, followed by separation of thelabelled target proteins, and sequencing. The protein kinase activity ofH2LAU20 may require incubation with a specific upstream effector. Thismay be achieved by preincubating H2LAU20 with lysates from a variety ofstimulated eukaryotic cells and ATP. These assays permit the discoveryand modification of compounds which inhibit H2LAU20 kinase activity invitro and would be expected to have effects on proliferation ofosteoblasts, chondrocytes, cardiac myocytes or skeletal myoblasts. Anyinhibitors so identified would be expected to have up-regulatory effectson proliferation and be useful as a therapeutic for the treatment andprevention of diseases such as osteoporosis, osteoarthritis,cardiomyopathy and cachexia.

This invention is also directed to the treatment and/or amelioration ofsuch diseases by administering an H2LAU20 inhibiting amount of acompound. Without wishing to be bound by any particular theory of thefunctioning of the H2LAU20 of this invention, among the usefulinhibitors of H2LAU20 function are those compounds which inhibit thekinase activity of the H2LAU20. Other sites of inhibition are, ofcourse, possibly owing to its position in a signal transduction cascade.Therefore, the present invention is also directed to inhibiting theinteraction of H2LAU20 with one or more of its upstream or downstreammodulators/substrates Inhibitors of protein-protein interactions betweenH2LAU20 and other factors could lead to the development ofpharmaceutical agents for the modulation of H2LAU20 activity.

The polynucleotides, polypeptides and antibodies to the polypeptide ofthe present invention may also be used to configure screening methodsfor detecting the effect of added compounds on the production of mRNAand polypeptide in cells. For example, an ELISA assay may be constructedfor measuring secreted or cell associated levels of polypeptide usingmonoclonal and polyclonal antibodies by standard methods known in theart. This can be used to discover agents which may inhibit or enhancethe production of polypeptide (also called antagonist or agonist,respectively) from suitably manipulated cells or tissues.

The polypeptide may be used to identify membrane bound or solublereceptors, if any, through standard receptor binding techniques known inthe art. These include, but are not limited to, ligand binding andcrosslinking assays in which the polypeptide islabeled with aradioactive isotope (for instance, ¹²⁵I), chemically modified (forinstance, biotinylated), or fused to a peptide sequence suitable fordetection or purification, and incubated with a source of the putativereceptor (cells, cell membranes, cell supernatants, tissue extracts,bodily fluids). Other methods include biophysical techniques such assurface plasmon resonance and spectroscopy. These screening methods mayalso be used to identify agonists and antagonists of the polypeptidewhich compete with the binding of the polypeptide to its receptors, ifany. Standard methods for conducting such assays are well understood inthe art.

Examples of potential polypeptide antagonists include antibodies or, insome cases, oligonucleotides or proteins which are closely related tothe ligands, substrates, receptors, enzymes, etc., as the case may be,of the polypeptide, e.g., a fragment of the ligands, substrates,receptors, enzymes, etc.; or small molecules which bind to thepolypeptide of the present invention but do not elicit a response, sothat the activity of the polypeptide is prevented.

Thus, in another aspect, the present invention relates to a screeningkit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for polypeptides of the present invention; orcompounds which decrease or enhance the production of such polypeptides,which comprises:

(a) a polypeptide of the present invention;

(b) a recombinant cell expressing a polypeptide of the presentinvention;

(c) a cell membrane expressing a polypeptide of the present invention;or

(d) antibody to a polypeptide of the present invention;

which polypeptide is preferably that of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

It will be readily appreciated by the skilled artisan that a polypeptideof the present invention may also be used in a method for thestructure-based design of an agonist, antagonist or inhibitor of thepolypeptide, by:

(a) determining in the first instance the three-dimensional structure ofthe polypeptide;

(b) deducing the three-dimensional structure for the likely reactive orbinding site(s) of an agonist, antagonist or inhibitor;

(c) synthesizing candidate compounds that are predicted to bind to orreact with the deduced binding or reactive site; and

(d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors.

It will be further appreciated that this will normally be an interactiveprocess.

In a further aspect, the present invention provides methods of treatingabnormal conditions such as, for instance, bone loss includingosteoporosis; inflammatory diseases such as Adult Respiratory DiseaseSyndrome (ARDS), Rheumatoid arthritis, Osteoarthritis, InflammatoryBowel Disease (IBD), psoriasis, dermatitis, asthma allergies; diabetesand associated disorders; infections such as bacterial, fungal,protozoan and viral infections, particularly infections caused by HIV-1or HIV-2; HIV-associated cachexia and other immunodeficiency disorders;septic shock; pain; injury; cancers including testicular cancer;anorexia: bulimia; Parkinson's disease; cardiovascular disease includingrestenosis, atherosclerosis, acute heart failure, myocardial infarction;hypotension: hypertension; urinary retention; angina pectoris; ulcers;benign prostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourette's syndrome, related to either an excess of, oran under-expression of, H2LAU20 polypeptide activity.

If the activity ofthe polypeptide is in excess, several approaches areavailable. One approach comprises administering to a subject in needthereof an inhibitor compound (antagonist) as hereinabove described,optionally in combination with a pharmaceutically acceptable carrier, inan amount effective to inhibit the function of the polypeptide, such as,for example, by blocking the binding of ligands, substrates, receptors,enzymes, etc., or by inhibiting a second signal, and thereby alleviatingthe abnormal condition. In another approach, soluble forms of thepolypeptides still capable of binding the ligand, substrate, enzymes,receptors, etc. in competition with endogenous polypeptide may beadministered. Typical examples of such competitors include fragments ofthe H2LAU20 polypeptide.

In still another approach, expression of the gene encoding endogenousH2LAU20 polypeptide can be inhibited using expression-blockingtechniques. Known such techniques involve the use of antisensesequences, either internally generated or separately administered (see,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides whichform triple helices with the gene can be supplied (see, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991)251:1360). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

For treating abnormal conditions related to an under-expressionofH2LAU20 and its activity, several approaches are also available. Oneapproach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates a polypeptide of thepresent invention, i.e., an agonist as described above, in combinationwith a pharmaceutically acceptable carrier, to thereby alleviate theabnormal condition. Alternatively, gene therapy may be employed toeffect the endogenous production of H2LAU20 by the relevant cells in thesubject. For example, a polynucleotide of the invention may beengineered for expression in a replication defective retroviral vector,as discussed above. The retroviral expression construct may then beisolated and introduced into a packaging cell transduced with aretroviral plasmid vector containing RNA encoding a polypeptide of thepresent invention such that the packaging cell now produces infectiousviral particles containing the gene of interest. These producer cellsmay be administered to a subject for engineering cells in vivo andexpression of the polypeptide in vivo. For an overview of gene therapy,see Chapter 20, Gene Therapy and other Molecular Genetic-basedTherapeutic Approaches, (and references cited therein) in HumanMolecular Genetics, T Strachan and A P Read, BIOS Scientific PublishersLtd (1996). Another approach is to administer a therapeutic amount of apolypeptide of the present invention in combination with a suitablepharmaceutical carrier.

In a further aspect, the present invention provides for pharmaceuticalcompositions comprising a therapeutically effective amount of apolypeptide, such as the soluble form of a polypeptide of the presentinvention, agonist/antagonist peptide or small molecule compound, incombination with a pharmaceutically acceptable carrier or excipient.Such carriers include, but are not limited to, saline, buffered saline,dextrose. water, glycerol, ethanol, and combinations thereof. Theinvention further relates to pharmaceutical packs and kits comprisingone or more containers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides and othercompounds of the present invention may be employed alone or inconjunction with other compounds, such as therapeutic compounds.

The composition will be adapted to the route of administration, forinstance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized. in the form of salves, pastes,gels, and the like.

The dosage range required depends on the choice of peptide or othercompounds of the present invention, the route of administration. thenature of the formulation, the nature of the subject's condition, andthe judgment of the attending practitioner. Suitable dosages, however,are in the range of 0.1-100 μg/kg of subject. Wide variations in theneeded dosage, however, are to be expected in view of the variety ofcompounds available and the differing efficiencies of various routes ofadministration. For example, oral administration would be expected torequire higher dosages than administration by intravenous injection.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in thesubject, in treatment modalities often referred to as “gene therapy” asdescribed above. Thus, for example, cells from a subject may beengineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example. by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

Polynucleotide and polypeptide sequences form a valuable informationresource with which to identiy further sequences of similar homology.This is most easily facilitated by storing the sequence in a computerreadable medium and then using the stored data to search a sequencedatabase using well known searching tools, such as GCC. Accordingly, ina further aspect, the present invention provides for a computer readablemedium having stored thereon a polynucleotide comprising the sequence ofSEQ ID NO:1 and/or a polypeptide sequence encoded thereby.

The following definitions are provided to facilitate understanding ofcertain terms used frequently hereinbefore.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of a Fab or other immunoglobulinexpression library.

“Isolated” means altered “by the hand of man” from the natural state. Ifan “isolated” composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not “isolated,” but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is “isolated”, as theterm is employed herein.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term “polynucleotide” also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well-described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination (see, for instance, PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993; Wold, F., Post-translational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors” , Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Post-translational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62).

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,and deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et at., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well-knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:

1)

Algorithm: Needleman and WVunsch. J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:

1)

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ D NO:1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO:1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90% 0.95 for95%, 0.97 for 97% or 100 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO:2, that is itmay be 100% identical, or it may include up to a certain integer numberof amino acid alterations as compared to the reference sequence suchthat the percent identity is less than 100% identity. Such alterationsare selected from the group consisting of at least one nucleic aciddeletion, substitution, including transition and transversion, orinsertion, and wherein said alterations may occur at the 5or 3′ terminalpositions of the reference polynucleotide sequence or anywhere betweenthose terminal positions, interspersed either individually among thenucleic acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of nucleic acidalterations for a given percent identity is determined by multiplyingthe total number of amino acids in SEQ ID NO:2 by the integer definingthe percent identity divided by 100 and then subtracting that productfrom said total number of amino acids in SEQ ID NO:2, or:

n _(n) ≦x _(n)−(x _(n) ·y),

wherein n_(n) is the number of amino acid alterations, x_(n) is thetotal number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for70%, 0.80 for 80%, 0.85 for 85% etc., · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 50, 60, 70, 80, 85, 90, 95,97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2,wherein said polypeptide sequence may be identical to the referencesequence of SEQ ID NO:2 or may include up to a certain integer number ofamino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the integer defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 100 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom xa.

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of SEQ ID NO:2, that is it may be100% identical, or it may include up to a certain integer number ofamino acid alterations as compared to the reference sequence such thatthe percent identity is less than 100% identity. Such alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for70%, 0.80 for 80%, 0.85 for 85% etc., and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

“Fusion protein” refers to a protein encoded by two, often unrelated,and fused genes or fragments thereof. In one example. EP-A-0 464discloses fusion proteins comprising various portions of constant regionof immunoglobulin molecules together with another human protein or partthereof. In many cases, employing an immunoglobulin Fc region as a partof a fusion protein is advantageous for use in therapy and diagnosisresulting in, for example, improved pharmacokinetic properties [see,e.g., EP-A 0232 262]. On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

SEQUENCE INFORMATION SEQ ID NO:1ATGGATGCTAAAAAGCCAAGGAAATGTGATTTGACTCCCTTCCTGGTTTTGAAAGCAAGAAAGAAACAAAAGTTCACCTCTGCGAAGGTTGGAAGTAAACTTTCGGTTCAGATCCAGAAGCCACCTTCCAATATCAAGAACTCCAGAATGACCCAAGTCTTTCATAAGAACACCAGTGTTACTTCACTCCCCTTTGTGGACACCAAGGGGAAGAAGAATACGGTAAGCTTCCCACACATTAGCAAGAAAGTCCTGCTGAAGTCATCCCTGCTGTATCAGGAGAATCAAGCTCACAATCAGATGCCGGCCTCAGAGCTCAAGGCTTCAGAAATACCTTTCCACCCTAGCATTAAAACCCAGGATCCCAAGGCAGAGGAGAAGTCACCAAAGAAGCAAAAGGTGACTCTGACAGCGGCAGAGGCCCTAAAGCTTTTTAAGAACCAGCTGTCTCCATATGAACAAAGTGAAATCCTGGGCTACGCGGAGCTGTGGTTCCTGGGTCTTGAAGCCAAGAAGCTCGACACGGCTCCTGAGAAATTTAGCAAGACGAGTTTTGATGATGAGCATGGCTTCTATCTGAAGGTCCTGCATGATCACATTGCCTACCGCTATGAAGTTCTGGAGACAATCGGGAAGGGGTCCTTTGGACAGGTGGCCAAGTGCTTGGATCACAAAAACAATGAGCTGGTGGCCCTGAAAATCATCAGGAACAAGAAGAGGTTTCACCAGCAGGCCCTGATGGAGCTGAAGATCCTGGAAGCTCTCAGAAAGAAGGACAAAGACAACACCTACAATGTGGTGCATATGAAGGACTTTTTCTACTTTCGCAATCACTTCTGCATCACCTTTGAGCTCCTGGGAATCAACTTGTATGAGTTGATGAAGAATAACAACTTTCAAGGCTTCAGTCTGTCCATAGTTCGGCGCTTCACTCTCTCTGTTTTGAAGTGCTTGCAGATGCTTTCGGTAGAGAAAATCATTCACTGTGATCTCAAGCCCGAAAATATAGTGCTATACCAAAAGGGCCAAGCCTCTGTTAAAGTCATTGACTTTGGATCAAGCTGTTATGAACACCAGAAAGTATACACGTACATCCAAAGCCGGTTCTACCGATCCCCAGAAGTGATCCTGGGCCACCCCTACGACGTGGCCATTGACATGTGGAGCCTGGGCTGCATCACGGCGGAGTTGTACACGGGCTACCCCCTGTTCCCCGGGGAGAATGAGGTGGAGCAGCTGGCCTGCATCATGGAGGTGCTGGGTCTGCCGCCAGCCGGCTTCATTCAGACAGCCTCCAGGAGACAGACATTCTTTGATTCCAAAGGTTTTCCTAAAAATATAACCAACAACAGGGGGAAAAAAAGATACCCAGATTCCAAGGACCTCACGATGGTGCTGAAAACCTATGACACCAGCTTCCTGGACTTTCTCAGAAGGTGTTTGGTATGGGAACCTTCTCTTCGCATGACCCCCGACCAGGCCCTCAAGCATGCTTGGATTCATCAGTCTCGGAACCTCAAGCCACAGCCCAGGCCCCAGACCCTGAGGAAATCCAATTCCTTTTTCCCCTCTGAGACAAGGAAGGACAAGGTTCAAGGCTGTCATCACTCGAGCAGAAAAGCAGATGAGATCACCAAAGAGACTACAGAGAAAACAAAAGATAGCCCCACGAAGCATGTTCAGCATTCAGGTGATCAGCAGGACTGTCTCCAGCACGGAGCTGACACTGTTCAGCTGCCTCAACTGGTAGACGCTCCCAAGAAGTCAGAGGCAGCTGTCGGGGCGGAGGTGTCCATGACCTCCCCAGGACAGAGCAAAAACTTCTCCCTCAAGAACACAAACGTTTTACCCCCTATTGTA TGA SEQ IDNO:2 MDAKKPRKCDLTPFLVLKARKKQKFTSAKVGSKLSVQIQKPPSNIKNSRMTQVFHKNTSVTSLPFVDTKGKKNTVSFPHISKKVLLKSSLLYQENQAHNQMPASELKASEIPFHPSIKTQDPKAEEKSPKKQKVTLTAAEALKLFKNQLSPYEQSEILGYAELWFLGLEAKKLDTAPEKFSKTSFDDEHGFYLKVLHDHIAYRYEVLETIGKGSFGQVAKCLDHKNNELVALKIIRNKKRFHQQALMELKILEALRKKDKDNTYNVVHMKDFFYFRNHFCITFELLGINLYELMKNNNFQGFSLSIVRRFTLSVLKCLQMLSVEKIIHCDLKPENIVLYQKGQASVKVIDFGSSCYEHQKVYTYIQSRFYRSPEVILGHPYDVAIDMWSLGCITAELYTGYPLFPGENEVEQLACIMEVLGLPPAGFIQTASRRQTFFDSKGFPKNITNNRGKKRYPDSKDLTMVLKTYDTSFLDFLRRCLVWEPSLRMTPDQALKLAWIHQSRNLKPQPRPQTLRKSNSFFPSETRKDKVQGCHHSSRKADEITKETTEKTKDSPTKHVQHSGDQQDCLQHGADTVQLPQLVDAPKKSEAAVGAEVSMTSPGQSKNFSLKNTNVLPPIV* SEQ ID NO:3   1 GGCACGAGGT GGCCATTGAC ATGTGGAGCCTGGGCTGCAT CACGGCGGAG  51 TTGTACACGG GCTACCCCCT GTTCCCCGGG GAGAATGAGGTGGAGCAGCT 101 GGCCTGCATC ATGGAGGTGC TGGGTCTGCC GCCAGCCGGC TTCATTCAGA151 CAGCCTCCAG GAGACAGACA TTCTTTGATT CCAAAGGTTT TCCTAAAAAT 201ATAACCAACA ACAGGGGGAA AAAAAGATAC CCAGATTCCA AGGACCTCAC 251 GATGGTGCTGAAAACCTATG ACACCAGCTT CCTGGACTTT CTCAGAAGGT 301 GTTTGGTATG GGAACCTTCTCTTCGCATGA CCCCGGACCA GGCCCTCAAG 351 CATGCTTGGA TTCATCAGTC TCGGAACCTCAAGCCACAGC CCAGGCCCCA 401 GACCCTGAGG AAATCCAATT CCTTTTTCCC CTCTGAGACAAGGAAgGACA 451 AGGTTCAAGG CTGTCATCaC TCGAGCAGAA AAGATGAGAT CACCAAAGAG501 ACTACAGAGA AAACAAAAGA TAGCCCCACG AAGCATGTTC AGCATTCAGG 551TGATCAGCAG GACTGTCTCC AGCACGGAGC TGACACTGTT CAGCTGCCTC 601 AACTGGTAGACGCTCCCAAG AAGTCAGAGG CAGCTGTCGG GGCGGAGGTG 651 TCCATGACCT CCCCAGGACAGAGCAAAAAC TTCTCCCTCA AGAACACAAA 701 CGTTTTACCC CCTATTGTAT GACCTTTGCTGAGGGTATGT CCTGCTCCTT 751 TCCACCAGTG ATTTGTATTA AGACAGCACT TATATTGTACAATACTTCAG 801 ACTGTTTTTT TTAAATACAT AAAACTTTAT GTTAAAAAAA AAAAAAAAAA851 A SEQ ID NO:4   1 HEVAIDMWSL GCITAELYTG YPLFPGENEV EQLACIMEVLGLPPAGFIQT  51 ASRRQTFFDS KGFPKNITNN RGKKRYPDSK DLTMVLKTYD TSFLDFLRRC101 LVWEPSLRMT PDQALKHAWI HQSRNLKPQP RPQTLRKSNS FFPSETRKDK 151VQGCHHSSRK DEITKETTEK TKDSPTKHVQ HSGDQQDCLQ HGADTVQLPQ 201 LVDAPKKSEAAVGAEVSMTS PGQSKNFSLK NTNVLPPIV

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence having at least 70% identity to the amino acid sequence of SEQID NO:2 over the entire length of SEQ ID NO:2, wherein said polypeptidehas H2LAU20 activity.
 2. An isolated polypeptide comprising the aminoacid sequence of SEQ ID NO:2.
 3. An isolated polypeptide which is theamino acid sequence of SEQ ID NO:2.